Methods and compositions for infusion of transiently engrafting, selected populations of allogeneic lymphocytes to treat cancer

ABSTRACT

The invention provides methods and compositions for administration of allogeneic lymphocytes as an exogenous source of CD4+ T cell help for endogenous, tumor-reactive CD8+ T cells. Depletion of CD8+ T cells from the donor lymphocyte infusion reduces the risk of sustained engraftment and graft-versus-host disease. Removal of regulatory T cells from the infused population may augment the ability of non-regulatory T cells to provide help for endogenous effectors of anti-tumor immunity. Allogeneic T cell therapy is typically given in the context of allogeneic stem cell transplantation, in which the patient receives highly immunosuppressive conditioning followed by an infusion of a stem cell graft containing unselected populations of mature T cells. In the treatment described here, the graft is engineered to minimize the possibility of sustained donor cell engraftment, and the anti-tumor effector T cells derive from the host.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. ApplicationSerial No. filed Nov. 3, 2014, now issued as U.S. Pat. No. 9,931,359;which is a 35 USC § 371 National Stage application of InternationalApplication No. PCT/US2013/032129 filed Mar. 15, 2013, now expired;which claims the benefit under 35 USC § 119(e) to U.S. Application Ser.No. 61/644,126 filed May 8, 2012, now expired. The disclosure of each ofthe prior applications is considered part of and is incorporated byreference in the disclosure of this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. IR01CA105148-01 and P01 CA15396 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates generally to immunology and more specifically, tomethods and compositions containing allogeneic lymphocytes to treatcancer.

Background Information

The immune system of a host provides the means for quickly andspecifically mounting a protective response to pathogenic microorganismsand also for contributing to rejection of malignant tumors. Immuneresponses have been generally described as including humoral responses,in which antibodies specific for antigens are produced by differentiatedB lymphocytes, and cell mediated responses, in which various types of Tlymphocytes eliminate antigens by a variety of mechanisms. For example,CD4 (also called CD4+) helper T cells that are capable of recognizingspecific antigens may respond by releasing soluble mediators such ascytokines to recruit additional cells of the immune system toparticipate in an immune response. CD8 (also called CD8+) cytotoxic Tcells are also capable of recognizing specific antigens and may bind toand destroy or damage an antigen-bearing cell or particle. Inparticular, cell mediated immune responses that include a cytotoxic Tlymphocyte (CTL) response can be important for elimination of tumorcells and cells infected by a microorganism, such as virus, bacteria, orparasite.

Cancer includes a broad range of diseases and affects approximately onein four individuals worldwide. A CTL response is a key feature ofeffective cancer vaccines; effective CD4 T cell help is also likely toplay a critical role in productive CD8 T cell activation and thusprovide clinical benefit.

With respect to microbial infections, malaria, tuberculosis, HIV-AIDSand other viral infections such as Herpes Simplex Virus (HSV) infections(the leading cause of genital ulcers worldwide) continue to contributeto global health concerns. HSV-2 prevalence is increasing at an alarmingrate across the globe. In the United States, the overall HSV-2 seroprevalence rate exceeds 20%, and in developing nations HSV-2 prevalenceis estimated between 30% and 50%. In addition to the profound burden ofHSV-2 infection in adults, the incidence of neonatal HSV-2 infection isincreasing. Even when treated, neonatal encephalitis from HSV-2infection has a mortality>15%, and the neurological morbidity amongHSV-2 infected infants is an additional 30 to 50% of surviving cases.Concomitant with the HSV-2 epidemic is a stark realization that HSV-2infection substantially increases the risk for HIV-1 acquisition andtransmission. Data from Africa show that HSV-2 infection can increasethe risk for HIV transmission by as much as 7-fold and that as many ashalf of newly acquired HIV cases are directly attributed to HSV-2infection. Overall, the relative risk of HIV acquisition increases morethan 2-fold in HSV-2-infected individuals.

Emerging evidence suggests that cancers induce a state ofunresponsiveness in lymphocytes that are specific for antigens uniquelyexpressed by the cancer. However, this unresponsiveness should be ableto be reversed. Several human tumors are infiltrated by CD8+ T cells,and the degree of CD8+ T cell infiltration often correlates with absenceof metastases and improved survival. However, these CD8+ T cells may noteliminate the cancer because of functional paralysis of tumor-specificCD4+ T cells.

SUMMARY OF THE INVENTION

The present invention is based on the seminal discovery that infusion ofallogeneic lymphocytes containing CD4+ T cells can break tolerance inhost anti-tumor CD8+ T cells, even though the donor cells do not engraftlong term in the recipient. Administration of chemotherapy prior to theallogeneic cell infusion can augment the anti-tumor effect of thetransiently engrafting lymphocytes, by promoting homeostatic expansionof the transferred lymphocytes, and/or by depleting host regulatory Tcells and myeloid-derived suppressor cells. The immune response tocancer is hampered by functional defects of the patient's CD4+ T cells.Infusions of allogeneic lymphocytes can provide an exogenous source ofCD4+ T cell help for endogenous, tumor-reactive CD8+ T cells. Depletionof CD8+ T cells from the donor lymphocyte infusion reduces the risk ofsustained engraftment and graft-versus-host disease. Removal ofregulatory T cells from the infused population may augment the abilityof non-regulatory T cells to provide help for endogenous effectors ofanti-tumor immunity.

Allogeneic T cell therapy is typically given in the context ofallogeneic stem cell transplantation, in which the patient receiveshighly immunosuppressive conditioning followed by an infusion of a stemcell graft containing unselected populations of mature T cells. The goalof alloSCT is to obtain sustained engraftment of the donor cells andentails the risk of mortality from graft-versus-host disease. In thetreatment described here, the graft is engineered to minimize thepossibility of sustained donor cell engraftment, and the anti-tumoreffector T cells derive from the host. Thus the therapy entails a uniquecooperation of host and donor lymphocytes during the period of transientdonor cell engraftment.

This is a treatment that can be applied to any human or animal cancer.Variations of the present invention include: 1) variations of thechemotherapy regimen that is given prior to infusion of allogeneiclymphocytes (may include cyclophosphamide, 5-fluorouracil, gemcitabine,dasatinib, combinations thereof; 2) variations in the source of donorlymphocytes (may be from related or unrelated donors, may includedefined mismatches at HLA Class I or Class II genetic loci; 3)variations in the types of cells selected for infusion, such asdepletion of CD4+CD25+ regulatory T cells, depletion of CD8+ T cells.Donors may be immunized to defined antigens prior to lymphocyteinfusions or may be polarized with cytokines ex vivo to enrich for TypeI (IFN-gamma producing) or Type 17 (IL-17-producing) T cells.

In a first embodiment, the invention provides a method of making anallogenic lymphocyte composition comprising: providing a peripheralblood cell composition from a human donor allogenic to the recipient,the peripheral blood cell composition comprising CD4+ T-cells, CD8+T-cells, and natural killer cells, wherein (i) the donor comprises atleast one human leukocyte antigen (HLA) Class II allele mismatchrelative to the recipient in the donor versus the recipient directionand the HLA Class II allele mismatch is at a gene selected from thegroup consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, and (ii) therecipient does not have detectable antibodies reactive against humanleukocyte antigens of the donor; and making the allogenic lymphocytecomposition from the peripheral blood cell composition by reducing thenumber of CD8+ T-cells in the peripheral blood cell composition by atleast one order of magnitude, wherein (a) the number of CD4+ T-cells inthe allogenic lymphocyte composition differs from the number of CD4+T-cells in the peripheral blood cell composition by less than about 50%,and (b) the number of natural killer cells in the allogenic lymphocytecomposition is less than or equal to the number of natural killer cellsin the peripheral blood cell composition, with the proviso that the CD4+T-cells of the allogenic lymphocyte composition are not activated exvivo.

In another embodiment, the invention provides a composition made by theinvention method. In yet another embodiment, the invention provides anallogenic human lymphocyte composition comprising: CD4+ T-cells andnatural killer cells from the peripheral blood cell composition of adonor, wherein (i) the donor comprises at least one human leukocyteantigen (HLA) Class II allele mismatch relative to the recipient in thedonor versus the recipient direction and the HLA Class II allelemismatch is at a gene selected from the group consisting of HLA-DRB1,HLA-DQB1, and HLA-DPB1, (ii) the recipient does not have detectableantibodies reactive against human leukocyte antigens of the donor, (iii)the number of CD4+ T-cells in the allogenic lymphocyte compositiondiffers from the number of CD4+ T-cells in the peripheral blood cellcomposition by less than about 50%, (iv) the number of donor CD4+T-cells based on an ideal body weight of the recipient in kilograms (kg)is between about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹ CD4+ T-cells/kg,(v) the number of natural killer cells in the allogenic lymphocytecomposition is less than or equal to the number of natural killer cellsin the peripheral blood cell composition, and (vi) the allogeniclymphocyte composition has at least one order of magnitude fewer CD8+T-cells relative to the peripheral blood cell composition; with theproviso that the CD4+ T-cells of the allogenic lymphocyte compositionare not activated ex vivo.

The invention also provides a method of treating a disease or conditionin a human subject, comprising: administering a lymphoreductivenon-lymphoablative treatment to the subject to induce transientlymphopenia in the subject; and subsequently administering to thesubject a first allogenic lymphocyte composition derived from aperipheral blood cell composition of a human, allogenic donor, the firstallogenic lymphocyte composition comprising a number of CD4+ T-cells anda number of natural killer cells from the peripheral blood cellcomposition of the donor, wherein (i) the donor comprises at least onehuman leukocyte antigen (HLA) Class II allele mismatch relative to thesubject in the donor versus the subject direction and the HLA Class IIallele mismatch is at a gene selected from the group consisting ofHLA-DRB1, HLA-DQB1, and HLA-DPB1, (ii) the subject does not havedetectable antibodies reactive against human leukocyte antigens of thedonor, (iii) the number of CD4+ T-cells in the first allogeniclymphocyte composition differs from the number of CD4+ T-cells in theperipheral blood cell composition by less than about 50%, (iv) thenumber of donor CD4+ T-cells based on an ideal body weight of thesubject in kilograms (kg) is between about 1×10⁵ CD4+ T-cells/kg andabout 1×10⁹ CD4+ T-cells/kg, (v) the number of natural killer cells inthe first allogenic lymphocyte composition is less than or equal to thenumber of natural killer cells in the peripheral blood cell composition,and (vi) the first allogenic lymphocyte composition has at least oneorder of magnitude fewer CD8+ T-cells relative to the peripheral bloodcell composition, with the proviso that the CD4+ T-cells of the firstallogenic lymphocyte composition are not activated ex vivo.

In one aspect, subsequent to administering the first allogeniclymphocyte composition to the subject, the method further comprisesadministering a successive lymphoreductive non-lymphoablative treatmentto the subject to induce transient lymphopenia in the subject; andsubsequently administering to the subject a successive allogeniclymphocyte composition derived from an additional peripheral blood cellcomposition of an additional human, allogenic donor, the successiveallogenic lymphocyte composition comprising a number of CD4+ T-cells anda number of natural killer cells from the additional peripheral bloodcell composition of the additional donor, wherein (i) the additionaldonor comprises at least one human leukocyte antigen (HLA) Class IIallele mismatch relative to the subject in the additional donor versusthe subject direction and the HLA Class II allele mismatch is at a geneselected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1,(ii) the subject does not have detectable antibodies reactive againsthuman leukocyte antigens of the additional donor, (iii) the number ofCD4+ T-cells in the successive allogenic lymphocyte composition differsfrom the number of CD4+ T-cells in the additional peripheral blood cellcomposition by less than about 50%, (iv) the number of additional donorCD4+ T-cells based on an ideal body weight of the subject in kilograms(kg) is between about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹ CD4+T-cells/kg, (v) the number of natural killer cells in the successiveallogenic lymphocyte composition is less than or equal to the number ofnatural killer cells in the additional peripheral blood cellcomposition, and (vi) the successive allogenic lymphocyte compositionhas at least one order of magnitude fewer CD8+ T-cells relative to theadditional peripheral blood cell composition.

The invention also provides a method of making an allogenic lymphocytecomposition for administration to a human recipient, comprising:providing a peripheral blood cell composition from a human donorallogenic to the recipient, the peripheral blood cell compositioncomprising a number of CD4+ T-cells, a number of CD8+ T-cells, and anumber of natural killer cells, wherein (i) the donor has CD4+ T-cellimmunity against an antigen present in the recipient, (ii) the donorcomprises at least one human leukocyte antigen (HLA) Class II allelematch relative to the recipient and the HLA Class II allele match is ata gene selected from the group consisting of HLA-DRB1, HLA-DQB1, andHLA-DPB1, and (iii) the recipient does not have detectable antibodiesreactive against human leukocyte antigens of the donor; and making theallogenic lymphocyte composition from the peripheral blood cellcomposition by reducing the number of CD8+ T-cells in the peripheralblood cell composition by at least one order of magnitude, wherein (a)the number of CD4+ T-cells in the allogenic lymphocyte compositiondiffers from the number of CD4+ T-cells in the peripheral blood cellcomposition by less than about 50%, and (b) the number of natural killercells in the allogenic lymphocyte composition is less than or equal tothe number of natural killer cells in the peripheral blood cellcomposition, with the proviso that the CD4+ T-cells of the allogeniclymphocyte composition are not activated ex vivo.

In another embodiment, the invention provides an allogenic lymphocytecomposition derived from a peripheral blood cell composition of a human,allogenic donor for administration to a human recipient, the allogeniclymphocyte composition comprising: a number of CD4+ T-cells and a numberof natural killer cells from the peripheral blood cell composition ofthe donor, wherein (i) the donor has CD4+ T-cell immunity against anantigen present in the recipient, (ii) the donor comprises at least onehuman leukocyte antigen (HLA) Class II allele match relative to therecipient and the HLA Class II allele match is at a gene selected fromthe group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) therecipient does not have detectable antibodies reactive against humanleukocyte antigens of the donor, (iv) the number of CD4+ T-cells in theallogenic lymphocyte composition differs from the number of CD4+ T-cellsin the peripheral blood cell composition by less than about 50%, (v) thenumber of donor CD4+ T-cells based on an ideal body weight of therecipient in kilograms (kg) is between about 1×105 CD4+ T-cells/kg andabout 1×109 CD4+ T-cells/kg, (vi) the number of natural killer cells inthe allogenic lymphocyte composition is less than or equal to the numberof natural killer cells in the peripheral blood cell composition, and(vii) the allogenic lymphocyte composition has at least one order ofmagnitude fewer CD8+ T-cells relative to the peripheral blood cellcomposition; with the proviso that the CD4+ T-cells of the allogeniclymphocyte composition are not activated ex vivo.

The invention also provides an allogenic lymphocyte composition derivedfrom a peripheral blood cell composition of a human, allogenic donor foradministration to a human recipient, the allogenic lymphocytecomposition comprising: a number of CD4+ T-cells and a number of naturalkiller cells from the peripheral blood cell composition of the donor,wherein (i) the donor has CD4+ T-cell immunity against an antigen notpresent in the recipient, (ii) the donor comprises at least one humanleukocyte antigen (HLA) Class II allele match relative to the recipientand the HLA Class II allele match is at a gene selected from the groupconsisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipient doesnot have detectable antibodies reactive against human leukocyte antigensof the donor, (iv) the number of CD4+ T-cells in the allogeniclymphocyte composition differs from the number of CD4+ T-cells in theperipheral blood cell composition by less than about 50%, (v) the numberof donor CD4+ T-cells based on an ideal body weight of the recipient inkilograms (kg) is between about 1×105 CD4+ T-cells/kg and about 1×109CD4+ T-cells/kg, (vi) the number of natural killer cells in theallogenic lymphocyte composition is less than or equal to the number ofnatural killer cells in the peripheral blood cell composition, and (vii)the allogenic lymphocyte composition has at least one order of magnitudefewer CD8+ T-cells relative to the peripheral blood cell composition;with the proviso that the CD4+ T-cells of the allogenic lymphocytecomposition are not activated ex vivo.

In one embodiment, the invention provides a kit for use in treating adisease or condition in a subject, the kit comprising: a lymphocytecomposition wherein the subject is the human recipient; and ananoparticle composition comprising nanoparticles comprising the antigennot present in the human recipient.

Further, there is a method of treating a disease or condition in a humansubject, comprising: administering to the subject an allogeniclymphocyte composition derived from a peripheral blood cell compositionof a human, allogenic donor, the allogenic lymphocyte compositioncomprising a number of CD4+ T-cells and a number of natural killer cellsfrom the peripheral blood cell composition of the donor, wherein (i) thedonor has CD4+ T-cell immunity against an antigen present in thesubject, (ii) the donor comprises at least one human leukocyte antigen(HLA) Class II allele match relative to the subject and the HLA Class IIallele match is at a gene selected from the group consisting ofHLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the subject does not havedetectable antibodies reactive against human leukocyte antigens of thedonor, (iv) the number of CD4+ T-cells in the allogenic lymphocytecomposition differs from the number of CD4+ T-cells in the peripheralblood cell composition by less than about 50%, (v) the number of donorCD4+ T-cells based on an ideal body weight of the subject in kilograms(kg) is between about 1×105 CD4+ T-cells/kg and about 1×109 CD4+T-cells/kg, (vi) the number of natural killer cells in the allogeniclymphocyte composition is less than or equal to the number of naturalkiller cells in the peripheral blood cell composition, and (vii) theallogenic lymphocyte composition has at least one order of magnitudefewer CD8+ T-cells relative to the peripheral blood cell composition;with the proviso that the CD4+ T-cells of the allogenic lymphocytecomposition are not activated ex vivo.

In one aspect, the invention provides a method of treating a disease orcondition in a human subject, comprising injecting a nanoparticlecomposition into a tumor, wherein the nanoparticle composition comprisesnanoparticles comprising an antigen not present in the subject, thusintroducing the antigen into the subject; administering to the subjectan allogenic lymphocyte composition derived from a peripheral blood cellcomposition of a human, allogenic donor, the allogenic lymphocytecomposition comprising a number of CD4+ T-cells and a number of naturalkiller cells from the peripheral blood cell composition of the donor,wherein (i) the donor has CD4+ T-cell immunity against the antigen, (ii)the donor comprises at least one human leukocyte antigen (HLA) Class IIallele match relative to the subject and the HLA Class II allele matchis at a gene selected from the group consisting of HLA-DRB1, HLA-DQB1,and HLA-DPB1, (iii) the subject does not have detectable antibodiesreactive against human leukocyte antigens of the donor, (iv) the numberof CD4+ T-cells in the allogenic lymphocyte composition differs from thenumber of CD4+ T-cells in the peripheral blood cell composition by lessthan about 50%, (v) the number of donor CD4+ T-cells based on an idealbody weight of the subject in kilograms (kg) is between about 1×105 CD4+T-cells/kg and about 1×109 CD4+ T-cells/kg, (vi) the number of naturalkiller cells in the allogenic lymphocyte composition is less than orequal to the number of natural killer cells in the peripheral blood cellcomposition, and (vii) the allogenic lymphocyte composition has at leastone order of magnitude fewer CD8+ T-cells relative to the peripheralblood cell composition; with the proviso that the CD4+ T-cells of theallogenic lymphocyte composition are not activated ex vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the hematocrits of the same five patients after BMT, withthe jagged portions reflecting the effect of transfusion (graph).

FIG. 2 shows non-engrafting DLI induces anti-tumor immunity (graph).

FIG. 3A shows engraftment of donor cells as vaccine plus alloCD4sprolong survival (graph). FIG. 3B shows a graph with donor CD4+ T cellchimerism and days post-transplantation.

FIG. 4 shows validation runs for CD8 depletion using leukapheresisproduct and phlebotomy specimens (flow cytometry results).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure arises at least in part from the seminaldiscovery that the immune response to cancer is hampered by functionaldefects of the patient's CD4+ T cells. Infusions of allogeneiclymphocytes can provide an exogenous source of CD4+ T cell help forendogenous, tumor-reactive CD8+ T cells. Depletion of CD8+ T cells fromthe donor lymphocyte infusion reduces the risk of sustained engraftmentand graft-versus-host disease. Removal of regulatory T cells from theinfused population may augment the ability of non-regulatory T cells toprovide help for endogenous effectors of anti-tumor immunity. AllogeneicT cell therapy is typically given in the context of allogeneic stem celltransplantation, in which the patient receives highly immunosuppressiveconditioning followed by an infusion of a stem cell graft containingunselected populations of mature T cells. In the treatment describedhere, the graft is engineered to minimize the possibility of sustaineddonor cell engraftment, and the anti-tumor effector T cells derive fromthe host. Thus, the therapy entails a unique cooperation of host anddonor lymphocytes during the period of transient donor cell engraftment.

In one embodiment, the invention provides a method of making anallogenic lymphocyte composition for administration to a humanrecipient, preferably, the donor and recipient are not the same human.The method includes providing a peripheral blood cell composition from ahuman donor allogenic to the recipient, the peripheral blood cellcomposition comprising a number of CD4+ T-cells, a number of CD8+T-cells, and a number of natural killer cells, some natural killer cellshave CD8+ antigen and may be removed by the “reducing” step; however,preferred lymphocyte compositions of the present invention comprise atleast some natural killer cells from the donor. In one aspect, (i) thedonor comprises at least one human leukocyte antigen (HLA) Class IIallele mismatch relative to the recipient in the donor versus therecipient direction (an HLA Class II allele mismatch in the donor versusrecipient direction, i.e., “graft-versus-host direction) and the HLAClass II allele mismatch is at a gene such as HLA-DRB1, HLA-DQB1, orHLA-DPB1. The recipient does not have detectable antibodies reactiveagainst human leukocyte antigens of the donor (”detectable antibodies”in this context are defined using standard methods of making thisdetermination (for example, the recipient does not have antibodiesagainst donor HLA molecules that are detectable by complement-dependentcytotoxicity, in flow cytometric cross-match assays a positive result isundesirable, or mean fluorescence intensity (WI) of 3000 or greater in asolid phase immunoassay is unacceptable).

The allogenic lymphocyte composition is made from the peripheral bloodcell composition by reducing the number of CD8+ T-cells in theperipheral blood cell composition by at least one order of magnitude,wherein (a) the number of CD4+ T-cells in the allogenic lymphocytecomposition differs from the number of CD4+ T-cells in the peripheralblood cell composition by less than about 50%. In a preferred embodimentthe ratio of the number of CD4+/the number of CD8+ T cells in thelymphocyte composition is preferably greater than or equal to about 30.Examples of some embodiments include, but are not limited to, thefollowing doses per kilogram of recipient ideal body weight: alymphocyte composition comprising 105 CD4+ cells typically has nogreater than 3.2×10³ CD8+ cells, 10⁶ CD4+ cells typically has no greaterthan 3.2×10⁴ CD8+ cells, 10⁷ CD4+ cells typically has no greater than3.2×10⁵ CD8+ cells, 10⁸ CD4+ cells typically has no greater than 3.2×10⁶CD8+ cells, and 5×10⁸ CD4+ cells typically has no greater than 1.6×10⁷CD8+ cells.

The number of natural killer cells in the allogenic lymphocytecomposition is less than or equal to the number of natural killer cellsin the peripheral blood cell composition, with the proviso that the CD4+T-cells of the allogenic lymphocyte composition are not activated exvivo, reduced by one order of magnitude, preferably about two orders ofmagnitude, more preferably to about five orders of magnitude. In oneembodiment, the number of CD8+ cells is reduced by about 2.5 orders ofmagnitude (e.g., using the magnetic bead cell sorter method).

In one aspect, wherein if the donor and recipient are ABO blood typeincompatible and the peripheral blood cell composition comprises anumber of red blood cells, then making the allogenic lymphocytecomposition further comprises reducing the number red blood cells. “ABOblood type incompatible,” as used herein, refers to when the recipienthas a major ABO red blood cell incompatibility against the donor, e.g.,the recipient is blood type O and the donor is blood type A, B, or AB,the recipient is type A and the donor is type B or AB, or the recipientis type B and the donor is type A or AB.

In one aspect, the number of red blood cells comprises less than orequal to about 50 ml in packed volume, e.g., less than or equal to about50 ml in packed volume, preferably less than or equal to about 30 ml inpacked volume, further “packed volume” should be defined, for example,centrifugation of the lymphocyte composition would result is a packedvolume of 50 ml or less of red blood cells; a measured volume sample ofthe lymphocyte composition could also be screened to provide aproportionally representative volume of packed blood cells.

In one aspect, the number of CD4+ T-cells in the allogenic lymphocytecomposition differs from the number of CD4+ T-cells in the peripheralblood cell composition by less than about 20%. In some embodiments, theCD4+ T-cells are less than about 50%, less than about 40%, preferablyless than about 20%, more preferably less than about 10%. In someembodiments, ex vivo expansion of CD4+ T-cells may be performed, in suchembodiments the number of CD4+ T-cells can greatly exceed the originalnumber. Such expansion is an alternative embodiment.

In some embodiments, CD4+ T-cells obtained from the donor are notintentionally expanded or intentionally differentiated ex vivo.Intentionally expanded or intentionally differentiated is distinguishedfrom expansion or differentiation of the CD4+ T-cells that is merely aside effect (not intentional, inadvertent) of the method, for example,CD4+ T-cells can sometimes undergo differentiation by coming intocontact with plastic, other examples of such inadvertent events. Inanother embodiment, there is a further proviso that stem cells have notbeen mobilized in the peripheral blood cell composition donor who isallogenic to the recipient.

In some aspects, reducing the CD8+ T-cells in the peripheral blood cellcomposition comprises using an anti-CD8+ antibody associated withmagnetic particles or an anti-CD8+ antibody plus complement. Theperipheral blood cell composition can be a whole blood product or anapheresis product, for example. Further, the HLA Class II allelemismatch in the donor versus the recipient direction can be a mismatchat HLA-DRB1. This limitation with an HLA Class II allele mismatch in thedonor versus recipient direction is for example “graft-versus-hostdirection”, wherein the at least one HLA Class II allele(s) mismatch inthe direction of the allogenic donor versus the recipient furthercomprises the same HLA Class II allele(s) mismatch between the allogenicdonor versus one or more first degree relatives of the recipient, whichis desirable to preserve the opportunity for bone marrow transplantationfrom the first degree relatives to the recipient; ideally, all of themismatches between donor versus recipient do not exist between apotential family bone marrow donor versus the recipient.

In some aspects, screening for one or more selection characteristic(s)is done and the screening is carried out on a subject selected from thegroup consisting the recipient, the donor, and one or more potentialallogenic donor(s). For example, a selection characteristic is screeningfor serological reactivity to an infectious agent antigen. An infectiousagent antigen is selected from the group consisting of a HumanImmunodeficiency Virus (HIV) antigen, a Hepatitis Virus antigen, and aCytomegalovirus antigen. Important agents to be screened for include,for example, HIV-1 antigen(s), HIV-2 antigen(s), hepatitis A virusantigen(s), hepatitis B virus antigen(s), hepatitis C virus antigen(s),CMV antigens, infectious diseases, etc. If the virus or infectious agentor an antigen thereof is the target of the therapy, one would not ruleout a donor having the desired CD4+ mediated immune response againstthat agent.

In one aspect, the infectious agent antigen is a Cytomegalovirusantigen, the recipient and the donor are screened, and there is noserological reactivity to the Cytomegalovirus antigen in the recipientor the donor. In one aspect, the viral antigen is an influenza antigenand the influenza antigen is a hemagglutinin antigen or a neuraminidaseantigen.

In another aspect, a selection characteristic is screening for more thanone HLA Class II alleles. In certain instances, a potential allogenicdonor is selected based on maximizing mismatch between the potentialallogenic donor versus the recipient, in the potential allogenic donorversus recipient direction, at the more than one HLA Class II alleles,and the potential allogenic donor is chosen as the donor. In certaininstances, a selection characteristic is screening for one or more HLAClass I allele(s).

A potential allogenic donor can be selected based on minimizing mismatchbetween the potential allogenic donor and the recipient at the more thanone HLA Class I allele(s), and the potential allogenic donor is chosenas the donor.

In one embodiment, the invention provides an allogenic lymphocytecomposition for administration to a human recipient obtained by themethod of the invention as described herein.

If the recipient is seropositive for the CMV antigen, then the status ofthe donor does not matter. In certain embodiments wherein the donor isnot immunized to an antigen that is present in, or will be delivered to,the recipient, the delivery of CD4+ T-cell help is contingent upon donorCD4+ T-cell recognition of allogenic HLA Class II molecules on therecipient's cells. An example of an “ideal donor” for the purpose ofexemplifying these embodiments of the present invention is thencompletely mismatched at HLA Class II alleles (in particular, HLA-DRB1,HLA-DQB1, and HLA-DPB1) and completely matched for Class I alleles (tomaximize survival of donor cells in the recipient and minimizealloantibody formation against Class I molecules). Further, the idealdonor is completely mismatched with unshared HLAs of first-degreerelatives of the recipient who are potential donors for allogenic stemcell transplantation.

In one embodiment, there is an allogenic lymphocyte composition derivedfrom a peripheral blood cell composition of a human, allogenic donor foradministration to a human recipient, the allogenic lymphocytecomposition comprising a number of CD4+ T-cells and a number of naturalkiller cells from the peripheral blood cell composition of the donor,wherein (i) the donor comprises at least one human leukocyte antigen(HLA) Class II allele mismatch relative to the recipient in the donorversus the recipient direction and the HLA Class II allele mismatch isat a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, andHLA-DPB1, (ii) the recipient does not have detectable antibodiesreactive against human leukocyte antigens of the donor, (iii) the numberof CD4+ T-cells in the allogenic lymphocyte composition differs from thenumber of CD4+ T-cells in the peripheral blood cell composition by lessthan about 50%, (including but not limited to less than about 50%, lessthan about 40%, preferably less than about 20%, more preferably lessthan about 10%). Further, “differs from the number of CD4+ T-cells inthe peripheral blood cell composition by less than about 50%” means plusor minus less than 50% of the number of CD4+ T-cells in the peripheralblood cell composition. For example, if the number of CD4+ T-cells inthe peripheral blood cell composition is 1×10⁵ CD4+ cells, then “differsfrom the number of CD4+ T-cells in the peripheral blood cell compositionby less than about 50%” means the number of CD4+ T-cells is between1.5×10⁵ and 0.5×10⁵. In the composition, the number of donor CD4+T-cells based on an ideal body weight (ideal body weight (IBW) is basedon height. For men, IBW=50+2.3 kg/inch over 5 feet. For women,IBW=45.5+2.3 kg/inch over 5 feet) of the recipient in kilograms (kg) isbetween about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹ CD4+ T-cells/kg (ina preferred embodiment, between about 1×10⁶ CD4+ T-cells/kg and about5×10⁸ CD4+ T-cells/kg); (v) the number of natural killer cells in theallogenic lymphocyte composition is less than or equal to the number ofnatural killer cells in the peripheral blood cell composition, and (vi)the allogenic lymphocyte composition has at least one order of magnitudefewer CD8+ T-cells relative to the peripheral blood cell composition;with the proviso that the CD4+ T-cells of the allogenic lymphocytecomposition are not activated ex vivo.

Also provided is a method of treating a disease or condition in a humansubject, comprising administering a lymphoreductive (in some embodimentsof this aspect of the present invention, it is desirable to provide alymphoreductive non-lymphoablative treatment to promote the homeostaticexpansion and differentiation of the administered lymphocytecomposition; in other embodiments it is desirable that the treatmentalso be myeloreductive (i.e., inhibiting or depleting suppressivemyeloid populations including myeloid-derived suppressor cells, tumorassociate macrophage, and or N2 neutrophils) non-lymphoablativetreatment to the subject to induce transient lymphopenia in the subject;and subsequently administering to the subject a first allogeniclymphocyte composition derived from a peripheral blood cell compositionof a human, allogenic donor, the first allogenic lymphocyte compositioncomprising a number of CD4+ T-cells and a number of natural killer cellsfrom the peripheral blood cell composition of the donor, wherein (i) thedonor comprises at least one human leukocyte antigen (HLA) Class IIallele mismatch relative to the subject in the donor versus the subjectdirection and the HLA Class II allele mismatch is at a gene selectedfrom the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (ii) thesubject does not have detectable antibodies reactive against humanleukocyte antigens of the donor, (iii) the number of CD4+ T-cells in thefirst allogenic lymphocyte composition differs from the number of CD4+T-cells in the peripheral blood cell composition by less than about 50%,(iv) the number of donor CD4+ T-cells based on an ideal body weight ofthe subject in kilograms (kg) is between about 1×10⁵ CD4+ T-cells/kg andabout 1×10⁹ CD4+ T-cells/kg, (v) the number of natural killer cells inthe first allogenic lymphocyte composition is less than or equal to thenumber of natural killer cells in the peripheral blood cell composition,and (vi) the first allogenic lymphocyte composition has at least oneorder of magnitude fewer CD8+ T-cells relative to the peripheral bloodcell composition, with the proviso that the CD4+ T-cells of the firstallogenic lymphocyte composition are not activated ex vivo.

While not wishing to be bound by a particular theory, the infused CD4+cells provide signals to other cell types, predominately the subject'sCD8+, macrophage, and/or antigen presenting cells that augment cytotoxicfunction of these cells in the subject (tolerized CD4+ of subject/exhausted CD8+ of subject); an example of treatment of a disease orcondition by this method should be exemplified, at least treatment ofmyelodysplastic syndrome.

In one aspect, the lymphoreductive non-lymphoablative treatmentcomprises treating the subject with one or more cytoreductive agentselected from the group consisting of alkylating agents, alkylsulphonates, nitrosoureas, triazenes, antimetabolites, pyrimidineanalogs, purine analogs, vinca alkaloids, epipodophyllotoxins,antibiotics, dibromomannitol, deoxyspergualine, dimethyl myleran andthiotepa. In one aspect, the lymphoreductive non-lymphoablativetreatment comprises treating the subject with an alkylating agent andthe alkylating agent is cyclophosphamide. In one aspect, subsequent toadministering the first allogenic lymphocyte composition to the subject,the method further comprises administration of an anti-tumor monoclonalantibody or anti-tumor monoclonal antibody/drug conjugate to thesubject.

In one aspect, kits are provided for use in treating a disease orcondition in a subject, the kit comprising: a lymphocyte composition asdescribed herein wherein the subject is the human recipient; and ananoparticle composition comprising nanoparticles comprising the antigennot present in the human recipient. In one aspect, the nanoparticlesfurther comprise a cytokine, for example, an interleukin or aninterferon. The cytokine can be an interleukin and is selected from thegroup consisting of IL-2, IL-7, IL-12, and IL-15. The cytokine is may bean interferon, e.g., interferon gamma, interferon beta, interferonalpha, interferon, tau, interferon omega, and consensus interferon.

The nanoparticles may further comprise a compound selected from thegroup consisting of a chemokine, an imaging agent, a photo antennamolecule, a thermal antenna molecule, and a Toll-like receptor ligand,ligands that promote differentiation of CD4+ T-cells into Type I (e.g.,IFN-gamma producing) CD4+ memory T-cells, ligands for receptors thatinduce activation of antigen presenting cells (e.g., anti-CD40antibodies or aptamers). Further, the nanoparticles may include an agentthat targets the nanoparticles to tumor cells or antigen-presentingcells.

In one aspect, method further comprises administration of the anti-tumormonoclonal antibody and the anti-tumor monoclonal antibody is selectedfrom the group consisting of rituximab, cetuximab, trastuzumab, andpertuzumab. In one aspect, the invention comprises administration of theanti-tumor monoclonal antibody/drug conjugate and the anti-tumormonoclonal antibody/drug conjugate is selected from the group consistingof brentuximab vedotin, gemtuzumab ozogamicin, trastuzumab emtansine,inotuzumab ozogamicin, glembatumumab vedotin, lorvotuzumab mertansine,cantuzumab mertansine, and milatuzumab-doxorubicin. In some aspects,admistration is first the allogenic lymphocyte composition and thenadministration of a chemotherapeutic agent to the subject. For example,the chemotherapeutic agent is selected from the group consisting ofdasatinib, nilotinib, ponatinib, imatinib, lapatinib, and vismodegib.

In one aspect, subsequent to administering the first allogeniclymphocyte composition to the subject, the method further comprisesadministration of a monoclonal antibody/CD4+ T-cell epitope conjugate tothe subject. In one aspect, subsequent to administering the firstallogenic lymphocyte composition to the subject, the method furthercomprises administering a successive lymphoreductive non-lymphoablativetreatment to the subject to induce transient lymphopenia in the subject;and subsequently administering to the subject a successive allogeniclymphocyte composition derived from an additional peripheral blood cellcomposition of an additional human, allogenic donor, the successiveallogenic lymphocyte composition comprising a number of CD4+ T-cells anda number of natural killer cells from the additional peripheral bloodcell composition of the additional donor, wherein (i) the additionaldonor comprises at least one human leukocyte antigen (HLA) Class IIallele mismatch relative to the subject in the additional donor versusthe subject direction and the HLA Class II allele mismatch is at a geneselected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1,(ii) the subject does not have detectable antibodies reactive againsthuman leukocyte antigens of the additional donor, (iii) the number ofCD4+ T-cells in the successive allogenic lymphocyte composition differsfrom the number of CD4+ T-cells in the additional peripheral blood cellcomposition by less than about 50%, (iv) the number of additional donorCD4+ T-cells based on an ideal body weight of the subject in kilograms(kg) is between about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹ CD4+T-cells/kg, (v) the number of natural killer cells in the successiveallogenic lymphocyte composition is less than or equal to the number ofnatural killer cells in the additional peripheral blood cellcomposition, and (vi) the successive allogenic lymphocyte compositionhas at least one order of magnitude fewer CD8+ T-cells relative to theadditional peripheral blood cell composition. With this method there isthe proviso that the CD4+ T-cells of the successive allogenic lymphocytecomposition are not activated ex vivo. Subsequent to administering thefirst allogenic lymphocyte composition to the subject, the methodfurther comprises administration of an agent that blocks negativesignaling in T-cells. The agent that blocks negative signaling in T-cellis selected from the group consisting of an anti-PD-1 antibody,ipilimumab, an anti-PD-L2 antibody, and a PD-1 fusion protein. Thedisease or condition is selected from the group consisting of a cancer,an autoimmune disorder, an organ transplantation, an allograftrejection, and a viral infection. For example, the disease or conditionis a cancer and the cancer is myelodysplastic syndrome.

In one embodiment, the invention provides a method of making anallogenic lymphocyte composition for administration to a humanrecipient, comprising providing a peripheral blood cell composition froma human donor allogenic to the recipient, the peripheral blood cellcomposition comprising a number of CD4+ T-cells, a number of CD8+T-cells, and a number of natural killer cells, wherein (i) the donor hasCD4+ T-cell immunity against an antigen present in the recipient, (ii)the donor comprises at least one human leukocyte antigen (HLA) Class IIallele match relative to the recipient and the HLA Class II allele matchis at a gene selected from the group consisting of HLA-DRB1, HLA-DQB1,and HLA-DPB1, and (iii) the recipient does not have detectableantibodies reactive against human leukocyte antigens of the donor; andmaking the allogenic lymphocyte composition from the peripheral bloodcell composition by reducing the number of CD8+ T-cells in theperipheral blood cell composition by at least one order of magnitude,wherein (a) the number of CD4+ T-cells in the allogenic lymphocytecomposition differs from the number of CD4+ T-cells in the peripheralblood cell composition by less than about 50%, and (b) the number ofnatural killer cells in the allogenic lymphocyte composition is lessthan or equal to the number of natural killer cells in the peripheralblood cell composition, with the proviso that the CD4+ T-cells of theallogenic lymphocyte composition are not activated ex vivo.

In one aspect, the antigen present in the recipient is selected from thegroup consisting of a neoplastic antigen (neoplastic antigen is anantigen associated with a neoplasm where neoplasm is defined as any newand abnormal cellular growth, specifically one in which cellularreplication is uncontrolled and progressive. Neoplasms may be benign,pre-malignant or malignant, and cancers are malignant neoplasms. Thusall cancer antigens are neoplastic antigens but not all neoplasticantigens are cancer antigens. Neoplastic idiotype (Id) is atumor-specific target, for example, in those B cell malignancies thatexpress this molecule on their cell surface, for example, lymphoma ormultiple myeloma, and include a viral antigen, a bacterial antigen, afungal antigen, a parasitic antigen, and a non-human animal antigen.

In one aspect, a potential allogenic donor is selected from the one ormore potential allogenic donor(s) based on minimizing mismatch betweenthe potential allogenic donor and the recipient at the more than one HLAClass II alleles, and the potential allogenic donor is chosen as thedonor. A potential allogenic donor is selected from the one or morepotential allogenic donor(s) based on minimizing mismatch between thepotential allogenic donor and the recipient at the one or more HLA ClassI allele(s), and the potential allogenic donor is chosen as the donor.

In one aspect, the antigen present in the recipient against which thedonor has immunity is a viral antigen and the viral antigen is selectedfrom the group consisting of a human papillomavirus antigen, an EpsteinBarr Virus antigen, a Kaposi's sarcoma-associated herpesvirus (KSHV)antigen, a Hepatitis A virus antigen, a Hepatitis B virus antigen, and aHepatitis C virus antigen. For example, the viral antigen is a humanpapillomavirus antigen and the human papillomavirus antigen is an E6 oran E7 antigenic peptide. In one aspect, the number of CD4+ T-cells inthe allogenic lymphocyte composition differs from the number of CD4+T-cells in the peripheral blood cell composition by less than about 20%.

In one embodiment is provided an allogenic lymphocyte compositionderived from a peripheral blood cell composition of a human, allogenicdonor for administration to a human recipient, the allogenic lymphocytecomposition comprising a number of CD4+ T-cells and a number of naturalkiller cells from the peripheral blood cell composition of the donor,wherein (i) the donor has CD4+ T-cell immunity against an antigenpresent in the recipient, (ii) the donor comprises at least one humanleukocyte antigen (HLA) Class II allele match relative to the recipientand the HLA Class II allele match is at a gene selected from the groupconsisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipient doesnot have detectable antibodies reactive against human leukocyte antigensof the donor, (iv) the number of CD4+ T-cells in the allogeniclymphocyte composition differs from the number of CD4+ T-cells in theperipheral blood cell composition by less than about 50%, (v) the numberof donor CD4+ T-cells based on an ideal body weight of the recipient inkilograms (kg) is between about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹CD4+ T-cells/kg, (vi) the number of natural killer cells in theallogenic lymphocyte composition is less than or equal to the number ofnatural killer cells in the peripheral blood cell composition, and (vii)the allogenic lymphocyte composition has at least one order of magnitudefewer CD8+ T-cells relative to the peripheral blood cell composition;with the proviso that the CD4+ T-cells of the allogenic lymphocytecomposition are not activated ex vivo.

The invention provides an allogenic lymphocyte composition derived froma peripheral blood cell composition of a human, allogenic donor foradministration to a human recipient, the allogenic lymphocytecomposition comprising a number of CD4+ T-cells and a number of naturalkiller cells from the peripheral blood cell composition of the donor,wherein (i) the donor has CD4+ T-cell immunity against an antigen notpresent in the recipient, (ii) the donor comprises at least one humanleukocyte antigen (HLA) Class II allele match relative to the recipientand the HLA Class II allele match is at a gene selected from the groupconsisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipient doesnot have detectable antibodies reactive against human leukocyte antigensof the donor, (iv) the number of CD4+ T-cells in the allogeniclymphocyte composition differs from the number of CD4+ T-cells in theperipheral blood cell composition by less than about 50%, (v) the numberof donor CD4+ T-cells based on an ideal body weight of the recipient inkilograms (kg) is between about 1×105 CD4+ T-cells/kg and about 1×109CD4+ T-cells/kg, (vi) the number of natural killer cells in theallogenic lymphocyte composition is less than or equal to the number ofnatural killer cells in the peripheral blood cell composition, and (vii)the allogenic lymphocyte composition has at least one order of magnitudefewer CD8+ T-cells relative to the peripheral blood cell composition;with the proviso that the CD4+ T-cells of the allogenic lymphocytecomposition are not activated ex vivo.

The invention provides a method of treating a disease or condition in ahuman subject, comprising administering to the subject an allogeniclymphocyte composition derived from a peripheral blood cell compositionof a human, allogenic donor, the allogenic lymphocyte compositioncomprising a number of CD4+ T-cells and a number of natural killer cellsfrom the peripheral blood cell composition of the donor, wherein (i) thedonor has CD4+ T-cell immunity against an antigen present in thesubject, (ii) the donor comprises at least one human leukocyte antigen(HLA) Class II allele match relative to the subject and the HLA Class IIallele match is at a gene selected from the group consisting ofHLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the subject does not havedetectable antibodies reactive against human leukocyte antigens of thedonor, (iv) the number of CD4+ T-cells in the allogenic lymphocytecomposition differs from the number of CD4+ T-cells in the peripheralblood cell composition by less than about 50%, (v) the number of donorCD4+ T-cells based on an ideal body weight of the subject in kilograms(kg) is between about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹ CD4+T-cells/kg, (vi) the number of natural killer cells in the allogeniclymphocyte composition is less than or equal to the number of naturalkiller cells in the peripheral blood cell composition, and (vii) theallogenic lymphocyte composition has at least one order of magnitudefewer CD8+ T-cells relative to the peripheral blood cell composition;with the proviso that the CD4+ T-cells of the allogenic lymphocytecomposition are not activated ex vivo.

The compositions and methods of the invention can be used against abroad range of cancers and tumor types, including but not limited tobladder cancer, brain cancer, breast cancer, colorectal cancer, cervicalcancer, gastrointestinal cancer, genitourinary cancer, head and neckcancer, lung cancer, ovarian cancer, prostate cancer, renal cancer, skincancer, and testicular cancer. More particularly, cancers that may betreated by the compositions and methods described herein include, butare not limited to, the following: cardiac cancers, including, forexample sarcoma, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma, andliposarcoma; myxoma; rhabdomyoma; fibroma; lipoma and teratoma; lungcancers, including, for example, bronchogenic carcinoma, e.g., squamouscell, undifferentiated small cell, undifferentiated large cell, andadenocarcinoma; alveolar and bronchiolar carcinoma; bronchial adenoma;sarcoma; lymphoma; chondromatous hamartoma; and mesothelioma;gastrointestinal cancer, including, for example, cancers of theesophagus, e.g., squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, and lymphoma; cancers of the stomach, e.g., carcinoma,lymphoma, and leiomyosarcoma; cancers of the pancreas, e.g., ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,and vipoma; cancers of the small bowel, e.g., adenocarcinoma, lymphoma,carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma,neurofibroma, and fibroma; cancers of the large bowel, e.g.,adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, andleiomyoma; genitourinary tract cancers, including, for example, cancersof the kidney, e.g., adenocarcinoma, Wilm's tumor (nephroblastoma),lymphoma, and leukemia; cancers of the bladder and urethra, e.g.,squamous cell carcinoma, transitional cell carcinoma, andadenocarcinoma; cancers of the prostate, e.g., adenocarcinoma, andsarcoma; cancer of the testis, e.g., seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, and limphoma;liver cancers, including, for example, hepatoma, e.g., hepatocellularcarcinoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma;hepatocellular adenoma; and hemangioma; bone cancers, including, forexample, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignantfibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignantlymphoma (reticulum cell sarcoma), multiple myeloma, malignant giantcell tumor chordoma, osteochrondroma (osteocartilaginous exostoses),benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteomaand giant cell tumors; nervous system cancers, including, for example,cancers of the skull, e.g., osteoma, hemangioma, granuloma, xanthoma,and osteitis defoinians; cancers of the meninges, e.g., meningioma,meningiosarcoma, and gliomatosis; cancers of the brain, e.g.,astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma),glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma,and congenital tumors; and cancers of the spinal cord, e.g.,neurofibroma, meningioma, glioma, and sarcoma; gynecological cancers,including, for example, cancers of the uterus, e.g., endometrialcarcinoma; cancers of the cervix, e.g., cervical carcinoma, and pretumor cervical dysplasia; cancers of the ovaries, e.g., ovariancarcinoma, including serous cystadenocarcinoma, mucinouscystadenocarcinoma, unclassified carcinoma, granulosa thecal celltumors, Sertoli Leydig cell tumors, dysgerminoma, and malignantteratoma; cancers of the vulva, e.g., squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, and melanoma;cancers of the vagina, e.g., clear cell carcinoma, squamous cellcarcinoma, botryoid sarcoma, and embryonal rhabdomyosarcoma; and cancersof the fallopian tubes, e.g., carcinoma; hematologic cancers, including,for example, cancers of the blood, e.g., acute myeloid leukemia, chronicmyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, andmyelodysplastic syndrome, Hodgkin's lymphoma, non Hodgkin's lymphoma(malignant lymphoma) and Waldenstrom's macroglobulinemia; skin cancers,including, for example, malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi,lipoma, angioma, dermatofibroma, keloids, psoriasis; and adrenal glandcancers, including, for example, neuroblastoma. In certain embodiments,when the disease is cancer, it may include a lung cancer tumor, a breastcancer tumor, a prostate cancer tumor, a brain cancer tumor, or a skincancer tumor for example.

The compositions of the invention can also be administered incombination with existing methods of treating cancers, for example bychemotherapy, irradiation, or surgery. Thus, there is further provided amethod of treating cancer comprising administering an effective amountof an invention composition to an individual in need of such treatment,wherein an effective amount of at least one further cancerchemotherapeutic agent is administered to the individual. Examples ofsuitable chemotherapeutic agents include any of: abarelix, aldesleukin,alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole,arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene,bleomycin, bortezombi, bortezomib, busulfan intravenous, busulfan oral,calusterone, capecitabine, carboplatin, carmustine, cetuximab,chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib,daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane,docetaxel, doxorubicin, dromostanolone propionate, eculizumab,epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide,exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine,fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumabozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan,idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a,irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin,leuprolide acetate, levamisole, lomustine, meclorethamine, megestrolacetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycinC, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine,nofetumomab, oxaliplatin, paclitaxel, pamidronate, panitumumab,pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin,pipobroman, plicamycin, procarbazine, quinacrine, rasburicase,rituximab, sorafenib, streptozocin, sunitinib, sunitinib maleate,tamoxifen, temozolomide, teniposide, testolactone, thalidomide,thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab,tretinoin, uracil mustard, valrubicin, vinblastine, vincristine,vinorelbine, vorinostat, and zoledronate.

In methods of the invention described herein, optionally beforeadministering to the subject the allogenic lymphocyte composition themethod further comprises administering a treatment to deplete or inhibitmyeloid-derived suppressor cells. The treatment to deplete or inhibitmyeloid-derived suppressor cells may comprise administration of a drugselected from the group consisting of dasatinib, 5-fluorouracil,taxotere, clodronate, and gemcitabine for example. Optionally, beforeadministering to the subject the allogenic lymphocyte composition themethod further comprises administering a treatment to deplete or inhibittumor associated macrophage cells. Optionally, before administering tothe subject the allogenic lymphocyte composition the method furthercomprises administering a treatment to deplete regulatory T cells. Thetreatment to deplete regulatory T cells may include administration of adrug selected from the group consisting of cyclophosphamide, denileukindiftitox, and daclizumab.

In another embodiment, the invention provides a method of treating adisease or condition in a human subject comprising injecting ananoparticle composition into a tumor (nanoparticles can be injectedinto or infused into the subject, wherein the nanoparticles furthercomprise a targeting agent, and the targeting agent binds to a targetcell such as a dispersed/non-localized neoplasm (e.g., a lymphoma orleukemia) where direct injection to all possible sites is not practicalor feasible), wherein the nanoparticle composition comprisesnanoparticles comprising an antigen not present in the subject, thusintroducing the antigen into the subject; administering to the subjectan allogenic lymphocyte composition derived from a peripheral blood cellcomposition of a human, allogenic donor, the allogenic lymphocytecomposition comprising a number of CD4+ T-cells and a number of naturalkiller cells from the peripheral blood cell composition of the donor,wherein (i) the donor has CD4+ T-cell immunity against the antigen, (ii)the donor comprises at least one human leukocyte antigen (HLA) Class IIallele match relative to the subject and the HLA Class II allele matchis at a gene selected from the group consisting of HLA-DRB1, HLA-DQB1,and HLA-DPB1, (iii) the subject does not have detectable antibodiesreactive against human leukocyte antigens of the donor, (iv) the numberof CD4+ T-cells in the allogenic lymphocyte composition differs from thenumber of CD4+ T-cells in the peripheral blood cell composition by lessthan about 50%, (v) the number of donor CD4+ T-cells based on an idealbody weight of the subject in kilograms (kg) is between about 1×10⁵ CD4+T-cells/kg and about 1×10⁹ CD4+ T-cells/kg, (vi) the number of naturalkiller cells in the allogenic lymphocyte composition is less than orequal to the number of natural killer cells in the peripheral blood cellcomposition, and (vii) the allogenic lymphocyte composition has at leastone order of magnitude fewer CD8+ T-cells relative to the peripheralblood cell composition; with the proviso that the CD4+ T-cells of theallogenic lymphocyte composition are not activated ex vivo. In oneaspect, the antigen is a non-human animal antigen and the non-humananimal antigen is a keyhole limpet hemocyanin antigen. In anotheraspect, the antigen is a viral antigen and the viral antigen is selectedfrom the group consisting of a human papillomavirus antigen, an EpsteinBarr Virus antigen, a Kaposi's sarcoma-associated herpesvirus (KSHV)antigen, a Hepatitis A virus antigen, a Hepatitis B virus antigen, and aHepatitis C virus antigen.

Compositions of the invention may be administered to the individual by avariety of routes, for example, orally, topically, parenterally,intravaginally, systemically, intramuscularly, rectally orintravenously. In certain embodiments, the composition is formulatedwith a pharmaceutical carrier. Preferably, the composition isadministered intravenously.

In some embodiments, the composition is combined with other anti-viralor anti-cancer therapies, such as administration of an anti-viral oranti-cancer agent, radiation therapy, phototherapy or immunotherapy. Theanti-viral or anti-cancer agent can be administered with an inventioncomposition either in the same formulation or in separate formulations,to enhance treatment. In these embodiments, the composition and theother therapies can be administered at the same time (simultaneously) orat separate times (sequentially), provided that they are administered insuch a manner and sufficiently close in time to have the desired effect.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES

The myelodysplastic syndromes (MDS) are a diverse group of malignantstem cell disorders characterized by dysplastic and ineffective bonemarrow production of blood cells and a variable risk of transformationto acute leukemia. These disorders may develop de novo or arise yearsafter exposure to potentially mutagenic chemotherapy.

Approximately 12,000-20,000 new cases of MDS will be diagnosed in theUnited States this year with a median age of onset of between 60 and 72.Current treatment outcomes for the myelodysplastic syndromes have beendisappointing. Age, performance status, and disease risk category, asdetermined by the International Prognostic Scoring System (IPSS),usually determine the choice of treatment modality. Patients<60 years ofage, who have good or excellent performance status and who are in theIPSS intermediate-2 or high risk categories, would predominantly beconsidered for high intensity therapies, since these IPSS categoriesconfer a median survival of 1.2 and 0.4 years, respectively.High-intensity therapies are defined as treatments requiringhospitalization, including intensive combination chemotherapy andhematopoetic cell transplantation.

Patients in the low or intermediate-1 category would generally beconsidered for low intensity therapies. These include treatments thatcan be administered in the outpatient clinic, such as hematopoeticgrowth factors, differentiation-inducing agents, biologic responsemodifiers, and low intensity chemotherapy. Patients with poorperformance status would be considered for supportive care or lowintensity therapies.

Example 1 IDE Device

The investigational agent to be used in this trial is the CliniMACSsystem with CliniMACS® CD8 reagent, a medical device that is used toenrich or deplete CD8+ T cells from human blood products. The CliniMACS®System intended for selection of CD8+ cells comprises four primarycomponents: 1) CliniMACS® CD8 Reagent—colloidal super paramagneticiron-dextran beads linked to a murine antibody against human CD8; 2)CliniMACSplus Instrument—a software controlled instrument that processesthe blood sample (cell product); 3) CliniMACS® Tubing Set, (Standard orLS)—a single-use, sterile, disposable tubing set with two proprietarycell selection columns; and 4) CliniMACS® PBS/EDTA Buffer—a sterile,isotonic phosphate buffered, 1 mM EDTA, saline solution, used asexternal wash and transport fluid for the in vitro preparation of bloodcells. The system utilizes magnetic cell sorting (MACS®), a powerfultool for the isolation of many cell types, to selectively enrich ordeplete the cell population of interest. In this case, CD8+ T cells arelabeled with a monoclonal antibody linked to super-paramagneticparticles and then are depleted from the blood product by passagethrough the CliniMACS system, which incorporates a strong permanentmagnet and a separation column with a ferromagnetic matrix to remove thelabeled cells. It is worth noting that the therapeutic agent in thistrial, CD8+ T cell-depleted blood cells, comes out of the device and isnot intended to contain any component of the device.

Example 2 Transiently Engrafting Donor Lymphocytes Induce Clinical TumorResponses

To date, only two therapies are capable of prolonging the survival ofpatients with MDS. The first, allogeneic BMT, has achieved some longterm cures, as well as delay in disease progression. This therapy isonly applicable to a small fraction of affected patients due to age,donor availability, and comorbidities. The second is themethyltransferase inhibitor, 5-azacitidine. This therapy has been shownto prolong median survival by 7 months compared to supportive carealone10. Some patients with MDS respond to immunosuppressive regimens,such as cyclosporine, antithymocyte globulin (ATG) or steroids, with asustained increase in blood counts. This finding is similar to aplasticanemia, where immunosuppression treats the autoimmune component leadingto the cytopenias. The favorable results obtained with agents thatspecifically target the immune system suggest that MDS is a disease thatis susceptible to immune modulation. One potential explanation for thebenefit of ATG, cyclosporine, and steroids is that these drugs unmaskthe activity of an endogenous anti-tumor immune response by selectivelyinhibiting or killing lymphocytes that suppress anti-tumor immunity.Further potential evidence for the existence of a cryptic, endogenousimmune response against MDS was seen in our trial of nonmyeloablative,partially HLA-mismatched (haploidentical) allogeneic bone marrowtransplantation, in which five patients experienced disease responsesdespite graft rejection. All five patients had at least transientreductions in the percentage of bone marrow blasts, and three of fivepatients, each of whom was dependent upon red blood cell+/−platelettransfusions prior to transplantation, became transfusion independent.Table 1 demonstrates that, despite absence of donor cell engraftment onday 30 after BMT, at least three of five patients had a reduction ofmarrow blasts lasting at least six months after BMT.

TABLE 1 Patient # Donor (age in chimerism Blast % Blast % Post years)Diagnosis (Day 30) Pre-BMT BMT (day) 1 (39) RAEB-t 0 22  0 (181+) 2 (62)AA → RAEB 0 15 0 (378) 3 (62) PCV → RAEB 0 8 0 (342) 4 (56) RAEB 0 5 2 5(59) RAEB-t 0 20 4 (73)  50 (78) 

FIG. 1 shows the hematocrits of the same five patients after BMT, withthe jagged portions reflecting the effect of transfusion. Three of fivepatients became transfusion independent, with patient #1 remaining inmorphologic and hematologic remission for at least three years. Moreinterestingly, patient #2 demonstrated a delayed hematologic response,becoming transfusion independent four months after BMT and three monthsafter documentation of graft rejection. In light of the sensitivity ofMDS to immunotherapy, we raise the possibility that an endogenous (i.e.,host-derived) anti-tumor immune response is reawakened by theimmunological perturbation provided by the transiently engrafting donorlymphocytes. This postulated mechanism, the awakening of an endogenousanti-response following graft rejection, may also account for theinduction of leukemia remission in patients receiving white blood celltransfusions after either no conditioning or only 100 cGy total bodyirradiation. Interestingly, in the latter study, three major clinicalresponses occurred in the absence of measurable donor chimerism.Clinical responses of both non-Hodgkin's and FIG. 2 shows a graph withpercent survival and days after tumor inoculation to show non-engraftingDLI induces anti-tumor immunity. BALB/c×C57BL/6 F1 mice (H-2b/d;=10/group) were conditioned with Cy 200 mg/kg IP on day −1. On day 0,they received 106 A20 lymphoma cells IV+/−5×107 spleen cells frompartially WIC-mismatched B6×C3H F1 donors (H-2b/k)+/−autologous tumorcell vaccine sc (106 irradiated A20+2×105 irr. B78H1-GM-CSF). Hodgkin'slymphoma despite the loss of donor chimerism following allogeneic BMThave also been described; in some of these cases, tumor regressionfollowing transiently engrafting donor lymphocyte infusions wasobserved. While it is certainly possible that donor T cells induced ananti-leukemic effect before they were rejected, it is also possible thata transient GVH reaction broke functional tolerance to leukemia in hostT cells. We have recently tested the hypothesis that transientlyengrafting donor lymphocyte infusions (DLI) can induce anti-tumor immuneresponses from host T cells in a mouse model. BALB/c×C57BL/6 F1 micewere treated with Cy on day −1, and on day 0 they received 106 A20lymphoma cells (of BALB/c origin) IV together with nothing,haploidentical DLI alone, autologous tumor cell vaccine alone, orDLI+vaccine. Compared to animals receiving either no treatment orvaccine alone after Cy conditioning, those that were conditioned with Cyand then treated with DLI alone or DLI+vaccine survived significantlylonger, with apparent cure achieved in five and four animals,respectively (FIG. 2). None of the nine cured animals had any detectabledonor chimerism when tested>100 days after DLI, suggesting that thedonor T cells were rejected. These results demonstrated that thecombination of Cy followed by partially MHC-mismatched DLI inducedsignificant anti-tumor effects. In order to characterize the role ofdonor CD4+ versus CD8+ T cells in the antitumor effect, the experimentwas repeated in recipients of Cy+vaccine+50 million mismatched spleencells that were either untreated or depleted of CD4+ T cells, CD8+ Tcells, or both. In this experiment, recipients of whole spleen DLI alldied of GVHD before day 20 (FIG. 3). In contrast, mice that receivedvaccine plus CD8+ T cell depleted spleen cells lived significantlylonger than mice receiving vaccine plus pan-T cell depleted spleen cells(p=0.04), indicating that depletion of CD8+ T cells abrogated lethalGVHD without abrogating anti-tumor immunity. In order to understand whydepletion of CD8+ T cells from the allogeneic DLI abrogated GVHD, westudied the survival of donor cells in mice conditioned with Cy and theninfused with mismatched spleen cells, either untreated or depleted ofeither or both T cell subsets. Interestingly, CD8+ T cell-depletedspleen cells engrafted only transiently, with donor CD4+ T cellchimerism peaking at 7 days after DLI and declining to undetectablelevels by day 21 (below). In contrast, sustained engraftment of donorcells was seen in all mice receiving DLI containing CD8+ T cells, andmost of these animals eventually died of GVHD. Taken together, theanimal studies demonstrate that Cy followed by CD8+ T cell depleted DLIinduces transient engraftment of donor cells and significant anti-tumoreffects without inducing acute GVHD. More recently, we have found thatdepletion of host CD8+ T cells prior to “Cy+DLI” significantlydiminishes the therapeutic effect, strongly implicating host CD8+ Tcells as critical mediators of the anti-tumor effect.

Example 3 Clinical Experience with CD8+ T Cell Depleted Allogeneic StemCell or Lymphocyte Infusions

There are no reports of patients treated with Cy followed by an infusionof CD8+ T cell depleted PBCs from haploidentical donors, so it is notpossible to provide preliminary safety data. However, there have beenreports of patients undergoing alloBMT who have received CD8+ Tcell-depleted grafts or of patients in relapse after alloBMT who havereceived CD8+ T cell depleted PBMC infusions. The goal of CD8+ T celldepletion was to reduce the incidence of GVHD while preserving theanti-leukemia effect of the infusion. With regard to GVHD, the studiesdid not yield a conclusive answer, with some showing a possible benefitand others showing none. Interestingly, infusion of CD8+ T cell-depletedDLI induced the activation of endogenous CD8+ T cells, a finding that isconsistent with the hypothesis that CD8+ T cell-depleted DLI caneffectively awaken a host CD8+ T cell response against cancer.

The results of two other studies are germane to considerations of thesafety of the proposed clinical trial. In the first study, patients withvarious hematologic malignancies received marrow from unrelated donorsthat were mismatched for either one HLA-DR allele or one HLA Class I(HLA-A or HLA-B) antigen. Patients received CD4+ T cell depleted graftscontaining titrated doses of CD8+ T cells. The major finding relevant tothe proposed study was that graft rejection occurred despitemyeloablative conditioning in six of ten patients receiving graftscontaining<3.1×10⁶ CD8+ T cells/kg of recipient body weight but in noneof fifteen patients receiving>3.1×10⁶ CD8+ T cells/kg. Thus, even aftermyeloablative conditioning, CD8+ T cell depletion significantlyincreases the risk of graft rejection, which nullifies the risk ofgraft-induced aplasia and GVHD. In the second study patients received Tcell-depleted, haploidentical peripheral blood stem cell (PBSC; n=15) orPBSC plus marrow grafts (n=28) containing a mean of 2.7×10⁴ or 3.5×10⁴CD3+ T cells/kg, respectively, which translates to CD8+ T cell doses ofapproximately 1-1.5×10⁴/kg. To facilitate engraftment in the face of Tcell depletion, patients were conditioned intensively and received“mega-dose” stem cell grafts containing a mean of 14.0×10⁶ or 10.6×10⁶CD34+ cells/kg for recipients of PBSC only versus PBSC plus marrow,respectively. In light of the low T cell content, no GVHD prophylaxiswas administered. Engraftment occurred in all 43 patients, and nopatient experienced acute or chronic GVHD as a result of thetransplantation procedure. These data demonstrate that even whensustained engraftment occurs in patients receiving myeloablativeconditioning, haploidentical grafts containing<10⁴ CD8+ T cells/kg areunlikely to cause GVHD. Since the device usually achieves>2 logdepletion of CD8+ T cells, the starting dose of CD8+ T cell depletedPBCs on the trial will likely contain fewer than 10⁴ CD8+ T cells/kg. Itis likely, then, that no patients receiving this dose will engraft orexperience GVHD, even if sustained engraftment occurs.

Example 4 CD8 Depletion Using the CliniMACS_System with CliniMACS-CD8Reagent

For this trial, CD8+ T cells will be depleted using the CliniMACS_systemwith the CliniMACS_CD8 Reagent (Miltenyi Biotec, Woburn, Mass.), underan investigator-sponsored IDE. We have performed three validation runs,the first using a leukapheresis product, and the last two usingphlebotomy specimens. Flow cytometry results from the last depletion areshown below, demonstrating excellent depletion of CD8+ T cells, from8.40% to 0.03% of total cells, and a corresponding enrichment of CD4+ Tcells and CD16+ or CD56+ NK cells (FIG. 4). Table 2 demonstrates allthree products meet the protocol criterion of having a CD8+ T cellnumber that is <3.2% of the CD4+ T cell number. Had products #2 and #3been used to deliver a dose of 106 CD4+ T cells/kg to a recipient withan ideal body weight of 70 kg, they would have contained 2.6×103 and7.7×102 CD8+ T cells/kg IBW, respectively, doses that are well below thethreshold for engraftment or GVHD induction. In comparison, the fivepatients who responded despite graft rejection received marrowscontaining a median of 1.43×10⁷ CD4+ T cells/kg (range 0.84-3.14×10⁷/kg)and 1.86×10⁷ CD8+ T cells/kg (range 0.43-2.16×10⁷/kg). Therefore, MDSpatients are capable of rejecting haploidentical cell infusionscontaining this many T cells.

TABLE 2 CE8 Depleted Cell Product Validation Results Total Total CD8+ %CD4+ CD4+ % CD8+ CD8+ Cells Log Percent Sterility Depletion Cells CellsCells Cells Depletion Viable Result #1 39.6% 7.6 × 10⁸  0.5% 9.5 × 10⁶1.95 98% Negative #2 11.6% 2.5 × 10⁸ 0.03% 6.5 × 10⁵ 2.43 90% Negative#3 39.9% 3.0 × 10⁸ 0.03% 2.3 × 10⁵ 2.67 97% Negative

Example 5 Correlative Laboratory Studies to Predict Response to Therapy

In light of the potential toxicities of immunosuppressive therapies,such as antithymocyte globulin, in patients with MDS35, numerousinvestigators have endeavored to identify patient characteristics thatcorrelate with response to therapy. Such characteristics that predictdisease response include hypocellular marrow, abnormal T cell receptorrepertoire by T cell receptor beta chain variable region CDR3 size byspectratype analysis, presence of cells with a phenotype characteristicof paroxysmal nocturnal hemoglobinuria (PNH), expression of HLADR15,trisomy 8, younger age, and shorter transfusion history. A major goal ofthis trial is to determine whether characteristics that predict responseto immunosuppressive therapy can also predict response to Cy+CD8+ Tcell-depleted haploidentical DLI. Patients entered onto this trial willhave examination of T cell receptor spectratype both before and aftertherapy. Cytogenetics and HLA typing will be performed routinely on allpatients.

Recent studies have uncovered a role for donor natural killer cellalloreactivity in preventing relapse of acute leukemia afterhaploidentical stem cell transplantation 36-38. More recently,alloreactive natural killer cells of donor origin have been found toprevent relapse of AML and MDS after HLA-identical stem celltransplantation39. These results underscore the need to characterize theexpressed repertoire of killer immunoglobulin-like receptors, or KIRs,on donor NK cells using both molecular and flow cytometric methods so asto identify donors expressing KIRs whose HLA ligands are missing onrecipient cells. These studies will be performed retrospectively in theImmunogenetics Laboratory and the results correlated with response totherapy.

Example 6 Rationale for the Proposed Trial Design

This is a standard phase I/II trial design that seeks to determine, inthe phase I portion of the trial, the maximally tolerated dose (MTD) ofCD8+ T cell-depleted haploidentical peripheral blood cells (CD8− PBCs)when infused after cyclophosphamide (Cy), and then to estimate, in thephase II portion of the trial, the efficacy of treatment with Cy plusthe MTD of CD8− PBCs. High dose Cy (>100 mg/kg) has been usedextensively as part of transplantation conditioning for patients withhematologic malignancies, and its safety is well-documented in thispopulation, including elderly patients (ages 55-66) with myelodysplasticsyndrome. The most serious risks of treatment, because they have thepotential to cause death, are prolonged aplasia and graft-versus hostdisease, both of which require sustained engraftment of the donor cells.The choice of the initial cell dose, 10⁵ CD4+ T cells/kg and <3.2×10³CD8+ T cells/kg, was based solely upon safety considerations.Specifically, grafts containing <104 CD8+ T cells/kg do not cause severeGVHD, even among patients receiving lethal conditioning and nopharmacologic immunosuppression after transplantation. Moreover, partialdepletion of CD8+ T cells from standard marrow grafts significantlyincreases the risk of graft rejection33, which is the desired outcome oftreatment in this trial. For these reasons, it is felt that a DLIproduct containing<104 CD8+ T cells/kg is unlikely to cause seriousadverse events.

Experience with haploidentical DLI, without CD8+ T-cell depletion, hasbeen recently published. In a phase I/II trial, 41 patients withrelapsed/refractory malignancies received nonablative conditioning with100 cGy total body irradiation, followed by infusion of 1×10⁶ to 2×108haploidentical CD3+ cells/kg, with 29 patients receiving the highestdose. Objective responses were achieved at the higher dose levels.Notably, 1×10⁸ CD3+ cells/dose was the minimum dose associated withresponse (25% response rate, or 2 of 8 patients), with 2×10⁸ CD3+cells/dose (the highest evaluated) associated with the greatest responserate (nearly 50%, in 10 of 21 patients). As proof of principle, allresponses occurred in the absence of sustained donor chimerism. In thehighest dose cohort, transient donor chimerism was seen but disappearedby 2 weeks in most patients, with one of two patients who converted tofull donor chimerism developing severe acute GVHD (steroid responsive,with subsequent development of fatal sepsis). An acute clinical syndrometermed “haplo immunostorm” likely secondary to cytokine flux(characterized by 1 or more of the following: fever, malaise, LFTabnormalities, rash and diarrhea) was seen commonly at the higher doselevels and was exquisitely responsive to steroids. This studydemonstrated the biological activity and manageable safety profile ofthis approach. The minimum CD3+ T-cell dose (not CD8+ depleted) requiredfor response in that study was 1×10⁸ cells/kg.

Example 7 Patient Selection

Patients must have pathologically confirmed: Myelodysplastic syndrome(MDS), IPSS score of Int-2 or high (see Appendix A for IPSS scoringsystem). Patients must have failed or be ineligible or intolerant oftreatment with 5-azacitidine.

Example 8 Treatment Plan

All patients will require documentation of a detailed history andphysical examination and standard evaluation of cardiac, liver and renalfunction, as designated in section 10. All patients will undergo a bonemarrow aspirate and biopsy for morphological, cytogenetic (ifapplicable) and flow cytometric (if applicable) evaluation no more thanone month prior to registration on protocol, along with other standarddisease evaluations (e.g., CT of chest, abdomen, pelvis) whereapplicable.

Pre-Treatment Evaluation

Cyclophosphamide will be administered as an iv infusion over 1-2 hr,(depending on volume) on days −2 and −1. The dose of cyclophosphamide is50 mg/kg/day. Dose is calculated based on the adjusted ideal body weightor actual body weight whichever is less. Body weight and height aremeasured directly. An approximate weight for height would be calculatedfrom a standard table or equations that reflect ideal “values”.

Cyclophosphamide and Pre-DLI Regimen

Patients will be instructed to increase fluids overnight beforecyclophosphamide administration. Hydration with normal saline at 3cc/kg/hr iv will be started 2 hr prior to cyclophosphamide, then therate will be reduced to 2 cc/kg/hr for 1 hr precyclophosphamide andcontinued for 8 hr post-cyclophosphamide. Mesna will be given in divideddoses iv 30 min pre- and at 3, 6, and 8 hr post cyclophosphamide.

Mesna dose will be based on the cyclophosphamide dose being given. Thetotal daily dose of mesna is equal to 80% of the total daily dose ofcyclophosphamide.

Prophylactic anti-microbial therapy will be started on Day 0 and willfollow institutional practice.

Antifungal prophylaxis will be administered as follows: Fluconazole 400mg po or IV qd, beginning Day 0 and continuing until the ANC is >500 for3 consecutive days (or for 2 consecutive measurements over a 3 dayperiod). Another appropriate prophylactic antifungal agent may besubstituted. Pneumocystis carinii pneumonia (PCP) prophylaxis will starton Day 0 and should continue until Day 60. Patients intolerant oftrimethoprim/sulfamethoxazole (Bactrim) will receive either dapsone orpentamidine as PCP prophylaxis. Viral prophylaxis will consist ofvalacyclovir or acyclovir from Day 0 to Day 60. An oral quinolone (e.g.,moxifloxacin or norfloxacin) will be administered according toinstitutional preference until the ANC is >500 for 3 consecutive days(or for 2 consecutive measurements over a 3 day period) following DLI.

All patients will receive infusion of haploidentical PBCs depleted ofCD8+ T cells using the CliniMACS® system with CliniMACS® CD8 reagent.The numbering of the dose levels is from the lowest to the highest celldose. The first cohort of patients (dose level 1) will receive Cy plusCD8+ T cell-depleted haploidentical PBCs (CD8− PBCs) containing anintended dose of 1×105 CD4+ T cells/kg of recipient IBW. If criteria fordose escalation are met, patients on dose level 2, 2b, 3, or 4 willreceive CD8− PBCs containing an intended dose of 1×10⁶, 3×106, 1×10⁷, or5×10⁷ CD4+ T cells/kg, respectively.

DLI Dose Calculation

The formula for calculating the volume of final (CD8-depleted) productthat will deliver the intended dose of CD4+ T cells is as follows:Intended volume (ml)=Intended CD4+ T cell dose (cells/kg)×RecipientIBW*(kg)/CD4+ T cell concentration (cells/ml)*Note—If actualweight<ideal, use actual weight.

However, the total number of CD8+ T cells that are infused may notexceed 3.2% of the intended number of CD4+ T cells to be infused (thenumerator of the equation above). If the ratio of CD4+/CD8+ cells in thedepleted product is less than 31.25 (=1/.032), then the volume of theproduct to be infused will be determined by the following formula: IfCD4/CD8 ratio of final product<31.25, then: Infused volume (ml)=Intendedvolume×(CD4/CD8 ratio)/31.25 If the ratio of CD4+/CD8+ cells in thedepleted product is equal to or greater than 31.25, then the volume ofthe product to be infused is the intended volume (formula 1): If CD4/CD8ratio of final product>31.25, then: Infused volume (ml)=Intended volume.

Transfusion Support

Platelet and packed red cell transfusions will be given per currentinstitutional recommendations.

Example 9 Duration of Therapy

Patients are eligible for only one lymphocyte infusion. This restrictionis in place because rejection of the infused lymphocytes is expected toinduce anamnestic immunity to cells of the donor or even to other closerelatives. Patient's peripheral blood will be obtained on day 60 andtested for the presence of human anti-mouse antibody (HAMA) and forcytotoxic antibodies against donor cells.

Duration of Follow-Up

Patients will be followed for a minimum of 60 days after DLI, and thenuntil death or disease progression, whichever occurs first. Patientsremoved from study for unacceptable adverse events or who developtreatment-related adverse events will be followed until resolution orstabilization of the adverse event.

Post DLI Monitoring

Patients remaining on study will have blood drawn on days 14, 28, and60, and six months after DLI. A CBC with manual differential will beobtained with these blood draws. Lymphocyte subsets, including thepercentage of cells expressing CD4 or CD8, will be analyzed by flowcytometry. After day 60, the patient will have monthly complete bloodcounts with white blood cell differential as long as there is nodocumented disease progression, until 6 months after DLI.

Disease Assessment

In addition to disease assessments specified above, results ofadditional disease assessments performed as standard of care will becollected for study purposes until death or disease progression,whichever occurs first.

Example 10 Dosing Delays/Dose Modification

Cyclophosphamide dose will not be modified. DLI dose will be modified inthe event of excessive content of CD8+ T cells.

Adverse Events: List and Reporting Requirements

The following information shall be collected on all patients with acuteGVHD: Date of onset (defined as the date of first biopsy confirmingGVHD) GVHD evaluation form at the time of onset, weekly until GVHDresolves, and Day 60 Initial overall clinical grade Maximum overallclinical grade Date of onset of grade III-IV acute GVHD, if any. Theoccurrence and severity of acute and chronic GVHD after Day 60 will becaptured at the patient's six month evaluation.

All instances of grade II-IV acute GVHD will be captured as adverseevents. Grade III-IV GVHD will be reported as a serious adverse event.

DLI-induced aplasia is defined as neutropenia (absolute neutrophilcount<500/ml) with any evidence of donor chimerism on day 60 or later.All cases of DLlinduced aplasia will be reported as serious adverseevents.

Example 11 Pharmaceutical Information Cyclophosphamide (Cytoxan®)

Cyclophosphamide is commercially available. Cyclophosphamide is analkylating agent which prevents cell division primarily by cross-linkingDNA strands. Cyclophosphamide is cell cycle non-specific.Cyclophosphamide for injection is available in 2000 mg vials which arereconstituted with 100 ml sterile water for injection. The concentrationof the reconstituted product is 20 mg/ml. The calculated dose will bediluted further in 250-500 ml of Dextrose 5% in water. Each dose will beinfused over 1-2 hr (depending on the total volume).

Clinical toxicities of cyclophosphamide include alopecia, nausea andvomiting, headache and dizziness, hemorrhagic cystitis, cardiotoxicity,immunosuppression, myelosuppression, pulmonary fibrosis, increasedhepatic enzymes and syndrome of inappropriate anti-diuretic hormone(SIADH). Cyclophosphamide will be dispensed by the Oncology Pharmacy andis produced by Mead Johnson Pharmaceuticals.

Mesna (Sodium-2-Mercapto Ethane Sulphonate)

Mesna is a prophylactic agent used to prevent hemorrhagic cystitisinduced by the oxasophosphorines (cyclophosphamide and ifosphamide). Ithas no intrinsic cytotoxicity and no antagonistic effects onchemotherapy. Mesna binds with acrolein, the urotoxic metaboliteproduced by the oxasophosphorines, to produce a non-toxic thioether andslows the rate of acrolein formation by combining with 4-hydroxymetabolites of oxasophosphorines.

Mesna is available in 200 mg, 400 mg and 1000 mg vials containing a 100mg/ml solution. Each dose of mesna will be diluted further in 50 ml ofnormal saline to be infused over 15 min. Mesna dose will be based on thecyclophosphamide dose being given. The total daily dose of mesna isequal to 80% of the total daily dose of cyclophosphamide.

At the doses used for uroprotection mesna is virtually non-toxic.However, adverse effects which may be attributable to mesna includenausea and vomiting, diarrhea, abdominal pain, altered taste, rash,urticaria, headache, joint or limb pain, hypotension and fatigue.

CBER IDE Device

Donors will have their blood collected via peripheral whole bloodcollection (450 ml into CPDA-1) or a leukapheresis procedure to collectperipheral white blood cells under steady state conditions (withoutmobilization). Each leukapheresis collection will be performed on acontinuous flow cell separator (COBE Spectra, Gambro) usinginstitutional standard operating procedures for lymphocyte collection.The method of blood donation, phlebotomy versus leukapheresis, will bedetermined by obtaining a peripheral blood absolute CD4+ T cell countwithin 30 days prior to donation and by estimating the volume of bloodrequired to obtain the targeted CD4+ T cell dose. Since the normal rangeof peripheral blood CD4+ T cell counts is 0.5-1.5×10⁶/ml, it is likelythat simple phlebotomy will be sufficient for dose levels 1-2, pheresismay be required for levels 2b but leukapheresis will be required fordose level 3 and 4.

Based upon extensive prior experience, a 4 hour leukapheresis procedureshould be sufficient to obtain 5×10⁷ CD4+ T cells/kg of recipient IBW.Target collections will be at least 30% more than the desired dose toaccommodate for cell loss during the depletion process.

The product will undergo CD8 depletion in the Graft EngineeringLaboratory. All standard operating procedures will be followed. Theproduct will be analyzed for nucleated cell count, CD3, CD4, CD8, CD16,and CD56. The product will be stored overnight and CD8 depletion willtake place on the CliniMACS® Selection System (Miltenyi Biotec, Auburn,Calif.). Prior to CD8 depletion, whole blood products will initially beprocessed to prepare a buffy coat concentrate and for major ABOincompatible donor/recipient pairs the buffy coat concentrate will befurther processed using lymphocyte separation medium to removecontaminating red blood cells. Processed whole blood products orapheresis products are then concentrated and resuspended in PBS/EDTAsupplemented with 0.5% human serum albumin.

Murine monoclonal CD8 antibody, conjugated to iron-dextransuper-paramagnetic particles is added and incubated at room temperaturefor 30 minutes. One vial of antibody will be used to treat up to 40×10⁹total white blood cells and up to 4×10⁹ CD8+ cells. Excess antibody willbe removed by washing 1 time and the product volume will be adjusted to100 ml with PBS/EDTA with albumin. It is then connected to the CliniMACSSelection System using a sterile disposable tubing set. The run isinitiated by a pre-set computer program which controls (i) the flow ofantibody-treated cells, (ii) washing that removes residual unboundcells, (iii) removal of the magnetic field around the column to releaseselected cells, and (iv) the final collection of CD8 depleted cells intoa bag. The entire process takes approximately 2-6 hours from completionof initial product concentration. The subsequent product will beanalyzed for cell count, viability, CD3, CD4, CD8, CD16, and CD56content. The CD4 concentration will be used to calculate the patientdose. The calculated volume will be removed and prepared for infusionaccording to institutional standard operating procedures.

Correlative/Special Studies Phenotypic Immune Reconstitution

Peripheral blood concentrations of lymphocyte subsets including CD4+ andCD8+ T cells will be determined using the absolute lymphocyte count andflow cytometry on days 14, 28, 60, and 6 months after DLI.

Analysis of Host CD8+ T Cell Repertoire Diversity by SpectratypeAnalysis. Recent studies indicate that the diversity of the T cellrepertoire can be assessed by T cell receptor V region spectratyping,which evaluates the CDR3/diversity/joining regions (Vβ-D-J-Cβ) of cellsexpressing a given V_gene. This region confers specificity of the T cellreceptor. Immunoscoping or V_spectratyping is remarkably useful forevaluating anti-tumor immune responses following therapy and immunereconstitution following bone marrow transplantation49. Moreover,spectratype analysis of MDS patients before and after immunosuppressivetherapy has revealed skewing of the T cell repertoire that normalizeswith a response to treatment. We therefore hypothesize that patientswith MDS and possibly CMML will have skewed T cell repertoires prior totreatment, that the DLI will initially induce a population ofalloreactive T cells, and that responders will eventually acquire anormal T cell repertoire as revealed by spectratype analysis.Pre-treatment CD8+ T cells will be obtained from patient peripheralblood mononuclear cells (PBMCs). To identify patient anti-donor reactiveT cells, pre-treatment PBMCs from the patient will be cultured for sevendays with irradiated donor PBMCs prior to cell sorting. The cultureperiod allow for the clonal expansion of patient anti-donor T cells.PBMCs will also be collected and CD8+ T cells will be purified on days14, 28, 60, and at six months.

Those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention. The presentinvention is not to be limited in scope by the specific embodimentsdescribed herein, which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description. Accordingly, the invention is limited only by thefollowing claims.

1-129. (canceled)
 130. A method of making an allogenic lymphocyte composition for administration to a human recipient, comprising: providing a peripheral blood cell composition from a human donor allogenic to the recipient, the peripheral blood cell composition comprising a number of CD4+ T-cells, a number of CD8+ T-cells, and a number of natural killer cells, wherein (i) the donor has CD4+ T-cell immunity against an antigen present in the recipient, (ii) the donor comprises at least one human leukocyte antigen (HLA) Class II allele match relative to the recipient and the HLA Class II allele match is at a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, and (iii) the recipient does not have detectable antibodies reactive against human leukocyte antigens of the donor; and making the allogenic lymphocyte composition from the peripheral blood cell composition by reducing the number of CD8+ T-cells in the peripheral blood cell composition by at least one order of magnitude, wherein (a) the number of CD4+ T-cells in the allogenic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%, and (b) the number of natural killer cells in the allogenic lymphocyte composition is less than or equal to the number of natural killer cells in the peripheral blood cell composition, with the proviso that the CD4+ T-cells of the allogenic lymphocyte composition are not activated ex vivo.
 131. The method of claim 130, wherein if the donor and recipient are ABO blood type incompatible and the peripheral blood cell composition comprises a number of red blood cells, then making the allogenic lymphocyte composition further comprises reducing the number red blood cells.
 132. The method of claim 131, wherein the number of red blood cells comprises less than or equal to about 50 ml in packed volume.
 133. The method of claim 130, wherein the antigen present in the recipient is selected from the group consisting of a neoplastic antigen, a neoplastic idiotype, a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, and a non-human animal antigen.
 134. The method of claim 130, wherein the antigen is a neoplastic antigen and the neoplastic antigen is a tumor antigen.
 135. The method of claim 130, wherein a subject selected from the group consisting of the recipient, the donor and one or more potential allogenic donor(s) has been screened for serological reactivity to an infectious agent antigen selected from the group consisting of a Human Immunodeficiency Virus (HIV) antigen, a Hepatitis Virus antigen, and a Cytomegalovirus antigen.
 136. The method of claim 130, wherein the antigen present in the recipient against which the donor has immunity is a viral antigen and the viral antigen is selected from the group consisting of a human papillomavirus antigen, an Epstein Barr Virus antigen, a Kaposi's sarcoma-associated herpesvirus (KSHV) antigen, a Hepatitis A virus antigen, a Hepatitis B virus antigen, and a Hepatitis C virus antigen.
 137. The method of claim 136, wherein the viral antigen is a human papillomavirus antigen and the human papillomavirus antigen is an E6 or an E7 antigenic peptide.
 138. The method of claim 130, wherein the number of CD4+ T-cells in the allogenic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 20%.
 139. The method of claim 130, with the further proviso that CD4+ T-cells obtained from the donor are not intentionally expanded or intentionally differentiated ex vivo.
 140. The method of claim 130, wherein reducing the CD8+ T-cells in the peripheral blood cell composition comprises using an anti-CD8+ antibody associated with magnetic particles or an anti-CD8+ antibody plus complement.
 141. An allogenic lymphocyte composition derived from a peripheral blood cell composition of a human, allogenic donor for administration to a human recipient, the allogenic lymphocyte composition comprising: a number of CD4+ T-cells and a number of natural killer cells from the peripheral blood cell composition of the donor, wherein (i) the donor has CD4+ T-cell immunity against an antigen present in the recipient, (ii) the donor comprises at least one human leukocyte antigen (HLA) Class II allele match relative to the recipient and the HLA Class II allele match is at a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipient does not have detectable antibodies reactive against human leukocyte antigens of the donor, (iv) the number of CD4+ T-cells in the allogenic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%, (v) the number of donor CD4+ T-cells based on an ideal body weight of the recipient in kilograms (kg) is between about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹ CD4+ T-cells/kg, (vi) the number of natural killer cells in the allogenic lymphocyte composition is less than or equal to the number of natural killer cells in the peripheral blood cell composition, and (vii) the allogenic lymphocyte composition has at least one order of magnitude fewer CD8+ T-cells relative to the peripheral blood cell composition; with the proviso that the CD4+ T-cells of the allogenic lymphocyte composition are not activated ex vivo.
 142. An allogenic lymphocyte composition derived from a peripheral blood cell composition of a human, allogenic donor for administration to a human recipient, the allogenic lymphocyte composition comprising: a number of CD4+ T-cells and a number of natural killer cells from the peripheral blood cell composition of the donor, wherein (i) the donor has CD4+ T-cell immunity against an antigen not present in the recipient, (ii) the donor comprises at least one human leukocyte antigen (HLA) Class II allele match relative to the recipient and the HLA Class II allele match is at a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the recipient does not have detectable antibodies reactive against human leukocyte antigens of the donor, (iv) the number of CD4+ T-cells in the allogenic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%, (v) the number of donor CD4+ T-cells based on an ideal body weight of the recipient in kilograms (kg) is between about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹ CD4+ T-cells/kg, (vi) the number of natural killer cells in the allogenic lymphocyte composition is less than or equal to the number of natural killer cells in the peripheral blood cell composition, and (vii) the allogenic lymphocyte composition has at least one order of magnitude fewer CD8+ T-cells relative to the peripheral blood cell composition; with the proviso that the CD4+ T-cells of the allogenic lymphocyte composition are not activated ex vivo.
 143. An allogenic lymphocyte composition derived from a peripheral blood cell composition of a human, allogenic donor, for use in a method of treating a disease or condition in a human subject, wherein: the allogenic lymphocyte composition comprising a number of CD4+ T-cells and a number of natural killer cells from the peripheral blood cell composition of the donor, wherein (i) the donor has CD4+ T-cell immunity against an antigen present in the subject, (ii) the donor comprises at least one human leukocyte antigen (HLA) Class II allele match relative to the subject and the HLA Class II allele match is at a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the subject does not have detectable antibodies reactive against human leukocyte antigens of the donor, (iv) the number of CD4+ T-cells in the allogenic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%, (v) the number of donor CD4+ T-cells based on an ideal body weight of the subject in kilograms (kg) is between about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹ CD4+ T-cells/kg, (vi) the number of natural killer cells in the allogenic lymphocyte composition is less than or equal to the number of natural killer cells in the peripheral blood cell composition, and (vii) the allogenic lymphocyte composition has at least one order of magnitude fewer CD8+ T-cells relative to the peripheral blood cell composition; with the proviso that the CD4+ T-cells of the allogenic lymphocyte composition are not activated ex vivo.
 144. An allogenic lymphocyte composition derived from a peripheral blood cell composition of a human, allogenic donor for use in a method of treating a disease or condition in a subject, wherein the allogenic lymphocyte composition comprises a number of CD4+ T-cells and a number of natural killer cells from the peripheral blood cell composition of the donor, wherein (i) the donor has CD4+ T-cell immunity against the antigen, (ii) the donor comprises at least one human leukocyte antigen (HLA) Class II allele match relative to the subject and the HLA Class II allele match is at a gene selected from the group consisting of HLA-DRB1, HLA-DQB1, and HLA-DPB1, (iii) the subject does not have detectable antibodies reactive against human leukocyte antigens of the donor, (iv) the number of CD4+ T-cells in the allogenic lymphocyte composition differs from the number of CD4+ T-cells in the peripheral blood cell composition by less than about 50%, (v) the number of donor CD4+ T-cells based on an ideal body weight of the subject in kilograms (kg) is between about 1×10⁵ CD4+ T-cells/kg and about 1×10⁹ CD4+ T-cells/kg, (vi) the number of natural killer cells in the allogenic lymphocyte composition is less than or equal to the number of natural killer cells in the peripheral blood cell composition, and (vii) the allogenic lymphocyte composition has at least one order of magnitude fewer CD8+ T-cells relative to the peripheral blood cell composition; with the proviso that the CD4+ T-cells of the allogenic lymphocyte composition are not activated ex vivo; and wherein the subject has been injected with a nanoparticle composition into a tumor, wherein the nanoparticle composition comprises nanoparticles comprising an antigen not present in the subject.
 145. An allogenic lymphocyte composition for use according to claim 143, wherein prior to administering to the subject the allogenic lymphocyte composition the method further comprises administering a lymphoreductive non-lymphoablative treatment to the subject to induce transient lymphopenia in the subject; administering a treatment to deplete or inhibit myeloid-derived suppressor cells; administering a treatment to deplete or inhibit tumor associated macrophage cells or administering a treatment to deplete regulatory T cells; or wherein subsequent to administering to the subject the allogenic lymphocyte composition the method further comprises administering a drug to induce selective depletion of alloreactive T-cells.
 146. An allogenic lymphocyte composition for use according to claim 145, wherein prior to administering to the subject the allogenic lymphocyte composition the treatment comprises administration of a drug selected from the group consisting of dasatinib, 5-fluorouracil, taxotere, clodronate, gemcitabine, cyclophosphamide, denileukin diftitox, and daclizumab.
 147. The lymphocyte composition of claim 141, wherein the antigen is selected from the group consisting of a neoplastic antigen, a neoplastic idiotype, a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, and a non-human animal antigen.
 148. The lymphocyte composition of claim 147, wherein the antigen is a viral antigen and the viral antigen is selected from the group consisting of a human papillomavirus antigen, an Epstein Barr Virus antigen, a Kaposi's sarcoma-associated herpesvirus (KSHV) antigen, a Hepatitis A virus antigen, a Hepatitis B virus antigen, a Hepatitis C virus antigen, and an influenza antigen.
 149. The lymphocyte composition of claim 147, wherein the viral antigen is a human papillomavirus antigen and the human papillomavirus antigen is an E6 or an E7 antigenic peptide, or wherein the viral antigen is an influenza antigen and the influenza antigen is a hemagglutinin antigen or a neuraminidase antigen.
 150. The lymphocyte composition of claim 141 wherein the allogenic lymphocyte composition is not immunologically reactive to a Cytomegalovirus antigen.
 151. The lymphocyte composition of claim 141 wherein the peripheral blood cell composition is a whole blood product or an apheresis product.
 152. The allogenic lymphocyte composition for use according to claim 149, wherein the nanoparticles further comprise a cytokine, wherein the cytokine is an interleukin and is selected from the group consisting of IL-2, IL-7, IL-12, and IL-15; or the cytokine is an interferon and is selected from the group consisting of interferon gamma, interferon beta, interferon alpha, interferon, tau, interferon omega, and consensus interferon. 